فهرست مطالب رضا پیله چیان لنگرودی

Clostridium is a broad genus of rod-shaped, anaerobic, gram-positive and spore-forming bacteria. It has 203 species and is classified into five isotypes (A, B, C, D, and E) based on four major toxins, iota (iA), alpha (cpa), beta (cpb) and epsilon (etx). These bacteria are one of the most important causes of disease in animals. Various toxins of these bacteria play an important role in pathogenesis. Newly discovered toxin such as NetB and TpeL could be mentioned. These toxins are involved in a variety of diseases, including necrotic enteritis. This article presents an overview of studies on NetB and TpeL toxins. Furthermore recent advances had been provided in the production of new generation vaccines. In this article, we demonstrated that the sequences of tpeL and netB were investigated using bioinformatics software for recombinant protein production. Different regions of the gene were selected and evaluated for fusion protein synthesis. The fusion gene was synthesized by PCR and the recombinant plasmid was transformed into E. coli. Then the fusion protein was purified by NI-NTA and used for confirmation by SDS-PAGE and Western blotting. Serum neutralization test was done to evaluate of antibody in rabbits. Mutants with less cytotoxic activity were also designed by site-directed mutagenesis kits and confirmed by sequencing. The results of this study suggested that new-generation vaccines could be used as a candidate vaccine against avian necrotic enteritis.

Clostridium novyi is endospore- forming, obligate anaerobic bacteria of the class clostridia. Cultivation of this bacterium in common culture is one of the problems with this bacterium. It is pathogenic, causing a wide variety of diseases in man and animals the spore of this bacterium has also received much attention in the treatment of cancer due to its ability to grow in anaerobic conditions inside solid tumors. In this study, the optimum growth conditions for vegetative and spore form was investigated. Bacterial culture was investigated in different solid media including BHI, Blood Agar, F medium and NP medium in vegetative form. Also, a suitable spore media was introduced, the spore purification method, an easy method for bacterial count and spore aspiration were investigated. The best rich media for C.novyi in vegetative form are F and NP media. That is, by adding horse blood to these media culture, the growth be lux but is not a sufficient factor for growth. There was an almost linear relationship between bacterial absorption and bacterial count for Clostridium novyi. Also, the difference in the number of spores germination which heating at 80 ° C for 15 minutes was significant.

Clostridium perfrinjens is an anaerobics, Gram-positive, rod-shaped and heat resistant bacterium of genus clostridium. C. perfringens is a spore-forming bacterium and widely occurring pathogen. The organism is grouped into 5 types (A, B, C, D, and E) on the basis of the production of 4 major toxins alpha, beta, epsilon, and iota toxins. Tpel Clostridium perfringens (C. perfringens) toxin have identified with A, B, and C types by cytotoxin activity in recent years. In this study C. perfringens type B had been used. Tpel caused to intestinal disease especially intestinal infections in human and necrotic enteritis in birds. In this study, perfect genomic DNA extracted by phenol-chloroform and Polymerase Chain Reaction (PCR) method used to isolation Tpel gene by a couple exclusive primer of perfect bacterium genomic DNA. PCR product after joining to pTZ57RL/T vector by TA cloning method in E. coli strain TOP10 susceptible became cloned and then colony PCR method used to screening transforming bacterium colonies with recombinant plasmid. Presence of fragment close to 2469bp on 1% agarose gel indicated that Tpel gene in E. coli strain TOP10 have be cloned.

Clostridium perfringens is cause of enterotoxaemia disease in sheep. The purpose of this research was to select the best strain Clostridium perfringens type D isolated from Iran and their comparison with vaccinal strain by MLD and potency tests. In this research 15 strains Clostridium perfringens type D were studied. At first, microbiological and biochemical tests were done then specimens were confirmed by PCR test. Finally, the best strain was selected by MLD test and the enterotoxaemia vaccine was prepared by the selected and the vaccine strain separately. The vaccines were kept for 45 days at refrigerator then potency test was done according to standard protocols. The results showed that the conditions for epsilon toxin production by isolated strain and the vaccine strain was nearly similar.

Iota-toxine est produite par le Clostridium perfringens de type E. Cette toxine provoque une entérotoxémie associée aux antibiotiques chez les agneaux et les veaux. Liota-toxine est une toxine binaire qui a deux composants dont Ia (le composant enzymatique) et Ib (le composant de liaison). Ib se lie au récepteur de surface des cellules cibles et translocation Ia dans le cytosol des cellules. Le but de cette recherche était le clonage de l'épitope toxigénique du gène iota a dans la souche d’E. coli Top10. Dans ce travail de recherche, la méthode phénol-chloroforme a été utilisée pour lextraction de lADN entier. L'épitope toxigénique de iota est un gène amplifié par PCR. Le produit de PCR a été ligaturé dans le clonage du vecteur pTZ57R / T et a été ensuite cloné dans E. colistrain Top10 selon la méthode de clonage TA,. L’analyse PCR des colonies a été utilisée pour cribler des colonies bactériennes qui ont été transformées avec des plasmides recombinants. La présence dun fragment de 446 pb sur legel dagarose a montré que l'épitope toxigénique de iota, un gène de C. perfringens, a été cloné dans la souche d’E. coli Top10.

Recombinant DNA technology is an in vitro molecular techniques to isolate and manipulate DNA fragments. Using this technique, construction chimeric molecules, called recombinant DNA molecules, then insert the recombinant molecules to living cells, replication and proliferation of the cells was possible. The fusion protein technology is the strategy to achieve rapid, cheap and efficient expression of proteins. Clostridium perfringens type D epsilon gene nucleotide sequence (etxD) and Clostridium septicum alpha gene (csa) were retrieved from GenBank. Then, to produce chimeric fusion protein, epsilon-alpha fusion gene was designed. Secondary and tertiary structures and characteristics of the fusion protein was determined by online software. The results showed that the designed fusion gene construction is suitable to expression of chimeric fusion protein.

Genus Clostridium، gram positive spore forming anaerobes are very important bacteria for human and animals pathogenesis. Clostridial diseases frequently occurs in Iranian farms and different Clostridium species are responsible for high mortality in domestic animals like as sheep and goats. In the present study، three groups of experimental vaccines were produced and compared to each others. The 1st group as bacterin pentavalent vaccine was produced in 20 liters glass vessels and adsorbed on aluminium hydroxide adjuvant. The 2nd group as bacterin pentavalent vaccine has been produced within in 12 liters fermenter and adsorbed on aluminium hydroxide adjuvant. Finally، the 3rd group as bacterin-toxoid pentavalent vaccine was produced in 12 liters fermenter and adsorbed on aluminium hydroxide adjuvant. Analysis for freedom from abnormal toxicity، safety and potency tests were performed for all produced vaccine groups. The results showed that the 3rd group of produced vaccine by fermenter and purified by ultrafiltration system is the best choice for scaling up.

Leptospirosis is an emerging infectious disease and is considered to be the most widespread zoonotic disease in the world. LigB is an immunogenic outer membrane protein. The leptospiral ligB gene expressed only in pathogenic Leptospira spp. The aim of this study was molecular diagnosis of pathogen Leptospires by PCR based on ligB gene. Five pathogenic Leptospires: L. canicola, L. grippotyphosa, L. pomona, L. icterohaemorrhagiae, L. serjoe hardjo and saprophytic L. biflexa were used in this study. The bacteria were inoculated into the selective culture medium and extraction of the genomic DNA was performed by standard Phenol-Chlorophorm method. The specific primers for proliferation of ligB gene were designed. The specificity and sensitivity of PCR method was evaluated. PCR product was 1041bp which indicated proliferation of ligB gene which was supported using electrophoresis. The PCR based on ligB gene detected all pathogenic reference serovars of Leptospira spp. tested. No PCR products were amplified from the non-pathogenic L. biflexa. Considering the spread of Leptosperosis in moderate and hot areas which have high rate of fall, a proper molecular diagnostic test with high specificity and sensitivity such as PCR is essential. PCR assay with high specificity and sensitivity may prove to be a rapid method for diagnosing acute leptospirosis. The results suggested that the PCR based on ligB gene can be used for detection of pathogenic leptospires.

Clostridium septicum a Gram positive anaerobic bacterium produces several toxins including alpha، beta، gamma and delta. C. septicum alpha toxin is lethal and is responsible for a serious disease known as gas gangrene. The aim of the present study was to molecular cloning and sequencing of C. septicum vaccine strain alpha toxin gene. Genomic DNA was extracted using standard phenol and chloroform extraction method، and the target gene was amplified through PCR by specific primers. Quality and quantity of PCR product was evaluated using agarose gel electrophoresis and confirmed with spectrophotometry. The PCR product was purified and was ligated in pJET1. 2blunt cloning vector and was used for E. coli/TOP10 competent cells transformation. pJETαsep recombinant plasmid was purified and sequenced using universal primers. Sequencing and BLAST analysis of csa showed over 99% identity to other previously deposited csa in the GenBank. The csa sequence was deposited in the GenBank under accession number JN793989. E. coli/TOP10/pJETαsep as a recombinant bacterium could be used for purifying of recombinant csa gene and its expression in the suitable prokaryotic hosts.

Braxy vaccine، a culture of a suitable strain or strains of Clostridium septicum in a fluid medium، its filtrate or derivatives، will be inactivated so that it produces its immunogenic activity without toxicity. Production of C. septicum vaccine (Braxy vaccine) using enriched culture media in fermenter. Conventional and enriched culture media were used for growing C. septicum (CN913) vaccinal strain in both 10 liter glass bottle and fermenter، in a triplicate manner. All twelve experiments were inspected to ensure compliance with the requirements of the vaccine international standards for minimum lethal dose (MLD)، sterility، freedom of abnormal toxicity، safety and potency tests. Results showed that the culture of C. septicum in fermenter using enriched culture media is the best. Based on international standards، 2. 5IU/mL was determined for C. septicum alpha toxin potency test، but in this study 4IU/mL was obtained. Using enriched culture media in fermenter is suitable for braxy vaccine production.

C. perfringens is an anaerobic gram positive spore forming bacterium that lives in soil، sediments and human and animal digestive tracks. This bacterium produces many toxins that are responsible for its virulence and are used for its classification. Based on the toxin production amount، C. perfringens is divided into five types A، B، C، D and E. 17 protein exotoxin is produced by this bacterium which 4 of them are major (alpha، beta، epsilon and Utah)، and others are minor toxins (sigma، theta، kappa، lambda، mu، nu، neuraminidase and enterotoxin). In 1993 C. perfringens chromosome was mapped for the first time. This map revealed details of the chromosome. In the recent years lot of studies in the field of Clostridium molecular biology and its DNA recombination has been done around the world and in Iran. This paper is a review of these studies.

Protein secretory systems are found in all living organisms including gram negative bacteria، and eukaryotic cell’s organelles that are derived from these bacteria. Unlike all other living organisms، gram negative bacteria have different protein secretory systems. At least there are four different independent secretory systems، which are responsible to transport the secreted proteins to the milieu or insert them into the bacterial cell surface. Comprising these systems in bacteria suggests that، beside the biogenic، mechanistic and evolutionary similarities، each acting independently. To display a protein or peptide with a distinct function at the surface of a living bacterial cell، is a challenging exercise with constantly increasing impact in many areas of biochemistry and biotechnology. Autodisplay system is developed based on autotransporters. This article introduces autodisplay protein expression based on autotransporters، on the bacterial cell surface، and their different potential applications and also in vaccine production.

The present work was carried out to investigate first, the ecology of the Sepedon flies as well as species of Lymnea snails and secondly, the biological effects of Sepedon fly larvae living on Lymnea. In this regard, the life cycle, death rate and compatibility of Lymnea species, as well as the life cycle of Sepedon flies in breeding cage and the activity of the new larvae in the same ecological condition were investigated. The results showed that Lymnea truncatula needs more sophisticated ecological and feeding conditions than other species of the snails. Lymnea stagnalis was found sensitive to temperature variation and this may lead to gradual or sudden death. Lymnea pregra and Lymnea palustris could well adapt with laboratory conditions. The killing effect (P<0.001) of the third-stage larvae on snails (3 to 4 weeks) was more than the effect of first- and second-stages larvae. This effect was more in a container with 2 cm water depth than a container with 5 cm depth.